Droplet Digital PCR Based Androgen Receptor Variant 7 (AR-V7) Detection from Prostate Cancer Patient Blood Biopsies

Abstract

Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

“…In total, 252 VCaP, 108 LNCap, and 186 PC-3 cells were evaluated and RCPs quantified by CellProfiler software. The in situ results reflect previously published data (22 ). VCaP cells seeded onto the CellCollector showed results comparable with cells on a slide (Fig.…”

“…1). The terms positive and negative refer to previously published data as detectable and not detectable mRNA, respectively (22 ). For VCaP cells, the average numbers of RCPs/ cell for AR-V7, AR-FL, and PSA were 3.2 (range, 0 -20), 2.7 (range, 0 -19), and 0.6 (range, 0 -7), respectively.…”

CTC-mRNA (AR-V7) Analysis from Blood Samples—Impact of Blood Collection Tube and Storage Time

“…Additionally, the detection of mRNA confirms that cells remain intact in the blood sample as it is well established that any released free RNA will be quickly degraded by RNases in the blood [27]. Since cultured 22Rv1 cells are potentially more robust and survive extended storage in blood samples better than CTCs, we confirmed that AR-V7 detection after 48 h storage was translatable to patient CTC samples by testing three patients previously shown to have high CTC AR-V7 expression [7]. Although a small decrease in AR-V7 levels after 48 h was detected in these patient samples, the decrease was marginal in this time frame and even in patient blood with low AR-V7, CTC derived AR-V7 was still detectable after 48 h blood storage at room temperature.…”

Improved Androgen Receptor Splice Variant 7 Detection Using a Highly Sensitive Assay to Predict Resistance to Abiraterone or Enzalutamide in Metastatic Prostate Cancer Patients

“…Several different blood-based AR-V7 detection assays are available, including CTC-based protein immunofluorescence and mRNA-based assays that can be performed using CTCs, whole-blood RNA, and plasma-derived exosomes [16,20]. Furthermore, different detection technologies are [11][12][13][14][15][21][22][23][24][25][26][27].…”